Pediatric Sarcoma Research Articles

Intra-abdominal desmoplastic small round cell tumor: The European pediatric Soft tissue sarcoma Study Group (EpSSG) experience.

This study describes the clinical findings of a consecutive series of pediatric and adolescent patients with a diagnosis of intra-abdominal desmoplastic small round cell tumor (DSRCT) prospectively enrolled in European pediatric Soft tissue sarcoma Study Group (EpSSG) protocols: the BERNIE study, the EpSSG MTS 2008 study, and the EpSSG NRSTS 2005 study. Patients aged less than 21years with a diagnosis of DSRCT arising in the abdomen were included. All trials recommended a multimodal approach including intensive multidrug chemotherapy and loco-regional treatment with surgery and/or radiotherapy whenever possible. The analysis included 32 cases (median age 13.7years, male:female ratio 1.5:1). Three patients had localized tumors, seven had regionally disseminated disease, and 22 extraperitoneal metastases. All but one patient received multidrug chemotherapy and 11 had maintenance chemotherapy. Loco-regional treatment consisted of surgery only in seven cases, surgery plus adjuvant radiotherapy in 10, and radiotherapy only in six. Among the 17 cases who had radiotherapy, six had irradiation of the primary site, 10 had whole abdominopelvic radiotherapy plus boost to macroscopic residual disease, and one had irradiation to lung metastases only. With a median follow-up of 76months (range: 18-124months), 5-year event-free and overall survivals were 19.7% and 21.0%, respectively. Event-free survival was significantly worse for patients who did not receive loco-regional treatment (p-value .007). The study confirmed that the outcome of patients with DSRCT remains dismal and did not improve over recent years despite an intensive multimodal treatment approach.

Surgical Outcomes in Paediatric Lower Limb Sarcomas: A Single Institutional Experience.

Primary bone sarcomas are rare tumours, comprising approximately 1-2% of adult and around 13-15% of all paediatric cancers. Limb salvage surgery is a challenge in complex musculoskeletal tumours. Young age adds to its complexity, since the growth potential of the reconstruct has to be considered to have optimal function of the salvaged limb. An observational retrospective study performed on 52 paediatric patients between Jan 2011 and Dec 2018 with malignant bone tumour of lower limbs had been assessed for postoperative functional outcomes with questionnaires and clinico-radiological examination for functional and oncological outcome. Out of 52 patients, 44 (85%) were diagnosed with osteosarcoma and the rest (15%) were diagnosed with Ewing sarcoma. Mean age was 12.88 years. Three-year overall survival rate was found to be 61.73%. Fifteen patients (28%) had metastasis at the time of presentation; non-metastasis patients (56.33 months) had a higher mean survival time, as compared to metastasis patient (29.06 months). Overall survival in group of patients undergoing biological, prosthesis and amputation surgeries were found to be similar. Mean MSTS score at final follow-up was 24.05 (80.1%), and in our study, 3.85% (2 out of 52) had local recurrence, and local recurrence free rate was found to be 96.15% (50 out of 52). The present study had excellent functional and oncological outcome at final follow-up which is comparable to the world literature. Complete surgical resection, if feasible, remains essential for cure.

The TLR3 L412F polymorphism prevents TLR3-mediated tumor cell death induction in pediatric sarcomas

The TLR3 L412F polymorphism prevents TLR3-mediated tumor cell death induction in pediatric sarcomas

Overall survival comparison between pediatric and adult Ewing sarcoma of bone and adult nomogram construction: a large population-based analysis.

Ewing sarcoma (ES) is a common primary bone tumor in children. Our study aimed to compare overall survival (OS) between pediatric and adult bone ES patients, identify independent prognostic factors and develop a nomogram for predicting OS in adult patients with ES of bone. We retrospectively analyzed data for the 2004-2015 period from the Surveillance, Epidemiology, and End Results (SEER) database. To guarantee well-balanced characteristics between the comparison groups, propensity score matching (PSM) was used. Kaplan-Meier (KM) curves were used to compare OS between pediatric and adult patients with ES of bone. Univariate and multivariate Cox regression analyses were used to screen independent prognostic factors for ES of bone, and a prognostic nomogram was constructed by using the factors identified. The prediction accuracy and clinical benefit were evaluated using receiver operating characteristic (ROC) curves, areas under the curves (AUCs), calibration curves, and decision curve analysis (DCA). Our results showed that adult ES patients had lower OS than younger ES patients. Age, surgery, chemotherapy, and TNM stage were independent risk factors for bone ES in adults and were used to develop a nomogram. AUCs for 3-, 5-, and 10-year OS were 76.4 (67.5, 85.3), 77.3 (68.6, 85.9) and 76.6 (68.6, 84.5), respectively. Calibration curves and DCA results indicated excellent performance for our nomogram. We found that ES pediatric patients have better OS than adult ES patients, and we constructed a practical nomogram to predict the 3-, 5- and 10-year OS of adult patients with ES of bone based on independent prognostic factors (age, surgery, chemotherapy, T stage, N stage and M stage).

The Pediatric Proton and Photon Therapy Comparison Cohort: Study Design for a Multicenter Retrospective Cohort to Investigate Subsequent Cancers After Pediatric Radiation Therapy

The physical properties of protons lower doses to surrounding normal tissues compared with photons, potentially reducing acute and long-term adverse effects, including subsequent cancers. The magnitude of benefit is uncertain, however, and currently based largely on modeling studies. Despite the paucity of directly comparative data, the number of proton centers and patients are expanding exponentially. Direct studies of the potential risks and benefits are needed in children, who have the highest risk of radiation-related subsequent cancers. The Pediatric Proton and Photon Therapy Comparison Cohort aims to meet this need. We are developing a record-linkage cohort of 10,000 proton and 10,000 photon therapy patients treated from 2007 to 2022 in the United States and Canada for pediatric central nervous system tumors, sarcomas, Hodgkin lymphoma, or neuroblastoma, the pediatric tumors most frequently treated with protons. Exposure assessment will be based on state-of-the-art dosimetry facilitated by collection of electronic radiation records for all eligible patients. Subsequent cancers and mortality will be ascertained by linkage to state and provincial cancer registries in the United States and Canada, respectively. The primary analysis will examine subsequent cancer risk after proton therapy compared with photon therapy, adjusting for potential confounders and accounting for competing risks. For the primary aim comparing overall subsequent cancer rates between proton and photon therapy, we estimated that with 10,000 patients in each treatment group there would be 80% power to detect a relative risk of 0.8 assuming a cumulative incidence of subsequent cancers of 2.5% by 15 years after diagnosis. To date, 9 institutions have joined the cohort and initiated data collection; additional centers will be added in the coming year(s). Our findings will affect clinical practice for pediatric patients with cancer by providing the first large-scale systematic comparison of the risk of subsequent cancers from proton compared with photon therapy.

The MYC-YBX1 Circuit in Maintaining Stem-like Vincristine-Resistant Cells in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma that causes significant devastation, with no effective therapy for relapsed disease. The mechanisms behind treatment failures are poorly understood. Our study showed that treatment of RMS cells with vincristine led to an increase in CD133-positive stem-like resistant cells. Single cell RNAseq analysis revealed that MYC and YBX1 were among the top-scoring transcription factors in CD133-high expressing cells. Targeting MYC and YBX1 using CRISPR/Cas9 reduced stem-like characteristics and viability of the vincristine-resistant cells. MYC and YBX1 showed mutual regulation, with MYC binding to the YBX1 promoter and YBX1 binding to MYC mRNA. The MYC inhibitor MYC361i synergized with vincristine to reduce tumor growth and stem-like cells in a zebrafish model of RMS. MYC and YBX expression showed a positive correlation in RMS patients, and high MYC expression correlated with poor survival. Targeting the MYC-YBX1 axis holds promise for improving survival in RMS patients.

Detection of circulating tumor-derived material in peripheral blood of pediatric sarcoma patients: A systematic review

BackgroundDetection of circulating tumor-derived material (cTM) in the peripheral blood (PB) of cancer patients has been shown to be useful in early diagnosis, prediction of prognosis, and disease monitoring. However, it has not yet been thoroughly evaluated for pediatric sarcoma patients. MethodsWe searched the PubMed and EMBASE databases for studies reporting the detection of circulating tumor cells, circulating tumor DNA, and circulating RNA in PB of pediatric sarcoma patients. Data on performance in identifying cTM and its applicability in diagnosis, and evaluation of tumor characteristics, prognostic factors, and treatment response was extracted from publications. ResultsA total of 79 studies were assigned for the present systematic review, including detection of circulating tumor cells (116 patients), circulating tumor DNA (716 patients), and circulating RNA (2887 patients). Circulating tumor cells were detected in 76% of patients. Circulating DNA was detected in 63% by targeted NGS, 66% by shallow WGS, and 79% by digital droplet PCR. Circulating RNA was detected in 37% of patients. ConclusionOf the cTM from Ewing's sarcoma and rhabdomyosarcoma ctDNA proved to be the best target for clinical application including diagnosis, tumor characterization, prognosis, and monitoring of disease progression and treatment response. For osteosarcoma the most promising targets are copy number alterations or patient specific micro RNAs, however, further investigations are needed to obtain consensus on clinical utility.

Prospects and Advances in Adoptive Natural Killer Cell Therapy for Unmet Therapeutic Needs in Pediatric Bone Sarcomas.

Malignant bone tumors are aggressive tumors, with a high tendency to metastasize, that are observed most frequently in adolescents during rapid growth spurts. Pediatric patients with malignant bone sarcomas, Ewing sarcoma and osteosarcoma, who present with progressive disease have dire survival rates despite aggressive therapy. These therapies can have long-term effects on bone growth, such as decreased bone mineral density and reduced longitudinal growth. New therapeutic approaches are therefore urgently needed for targeting pediatric malignant bone tumors. Harnessing the power of the immune system against cancer has improved the survival rates dramatically in certain cancer types. Natural killer (NK) cells are a heterogeneous group of innate effector cells that possess numerous antitumor effects, such as cytolysis and cytokine production. Pediatric sarcoma cells have been shown to be especially susceptible to NK-cell-mediated killing. NK-cell adoptive therapy confers numerous advantages over T-cell adoptive therapy, including a good safety profile and a lack of major histocompatibility complex restriction. NK-cell immunotherapy has the potential to be a new therapy for pediatric malignant bone tumors. In this manuscript, we review the general characteristics of osteosarcoma and Ewing sarcoma, discuss the long-term effects of sarcoma treatment on bones, and the barriers to effective immunotherapy in bone sarcomas. We then present the laboratory and clinical studies on NK-cell immunotherapy for pediatric malignant bone tumors. We discuss the various donor sources and NK-cell types, the engineering of NK cells and combinatorial treatment approaches that are being studied to overcome the current challenges in adoptive NK-cell therapy, while suggesting approaches for future studies on NK-cell immunotherapy in pediatric bone tumors.

Metastatic adult-type non-rhabdomyosarcoma soft tissue sarcomas in children and adolescents: A cohort study from the European paediatric Soft tissue sarcoma Study Group.

Limited data exist on the clinical behavior of pediatric non-rhabdomyosarcoma soft tissue sarcomas (NRSTS) with distant metastases at onset, and a clear standard of care has not yet been defined. This cohort study reports on pediatric adult-type metastatic NRSTS enrolled in two concurrent prospective European studies, i.e., the randomized BERNIE study and the single-arm MTS 2008 study developed by the European paediatric Soft tissue sarcoma Study Group. Treatment programs were originally designed for patients with metastatic rhabdomyosarcoma, i.e., nine courses of multidrug chemotherapy (with or without bevacizumab in the BERNIE study), followed by 12 cycles of maintenance therapy, whereas radiotherapy and/or surgery (on primary tumor and/or metastases) were delayed until after seven courses of chemotherapy had been administered. The study included 61 patients <21years old treated from July 2008 to December 2016. The lung was the site of metastases in 75% of the cases. All patients received multi-agent chemotherapy, 44% had local therapy to primary tumor, and 18% had treatment of metastases. Median time to progression/relapse was 6months. A high rate of tumor progression was observed during the initial part of the chemotherapy program. With a median follow-up of 41.5months (range, 2-111months), 3-year event-free survival and overall survival were 15.4% (95% confidence interval [CI], 7.6-25.7) and 34.9% (95% CI, 22.7-47.5), respectively. There were no statistically significant differences in outcome depending on the type of treatment administered. The study confirmed the overall poor outcome for patients with metastatic NRSTS, whose treatment remains a challenge. Pediatric non-rhabdomyosarcoma soft tissue sarcomas form a heterogeneous group of rare tumors. Although recent international studies have defined the standard of care for patients with localized disease, limited data are available on the clinical behavior of patients with distant metastases. This study on 61 metastatic cases treated on two prospective European protocols confirms that the chances of survival of such patients are often dismal and a standard treatment is still lacking.

Heat Shock Proteins: Important Helpers for the Development, Maintenance and Regeneration of Skeletal Muscles

The skeletal muscle is a highly plastic tissue that shows a remarkable adaptive capacity in response to acute and resistance exercise, and modifies its composition to adapt to use and disuse, a process referred to as muscle plasticity. Heat shock proteins (HSPs), a class of evolutionarily conserved molecular chaperones, have been implicated in the regulation of skeletal muscle plasticity. Here, we summarize key findings supporting the notion that HSPs are important components required to maintain skeletal muscle integrity and functionality. HSPs participate in the transcriptional program required for myogenesis and are activated following muscle exercise and injury. Their dysfunction, either as a consequence of improper expression or genetic mutations, contributes to muscle atrophy and leads to the development of myopathies and peripheral motor neuropathies. Denervation/reinnervation and repeated rounds of nerve degeneration/regeneration have been observed in motor neuropathies, suggesting that an imbalance in HSP expression and function may impair the repair of the neuromuscular junctions. Boosting HSP activity may help preventing muscle atrophy by promoting muscle differentiation and helping the repair of NMJs. Boosting HSP function may also help to combat the development of rhabdomyosarcoma (RMS), a highly aggressive type of pediatric soft tissue sarcoma whose cells have skeletal muscle features but are unable to fully differentiate into skeletal muscle cells.

Switchable CAR T cell strategy against osteosarcoma

Immunotherapy with chimeric antigen receptor T (CAR T) cells has changed the treatment of hematological malignances, but they are still a challenge for solid tumors, including pediatric sarcomas. Here, we report a switchable CAR T cell strategy based on anti-FITC CAR T cells and a switch molecule conjugated with FITC for targeting osteosarcoma (OS) tumors. As a potential target, we analyzed the expression of B7-H3, an immune checkpoint inhibitor, in OS cell lines. In addition, we evaluate the capacity of an anti-B7-H3 monoclonal antibody conjugated with FITC (anti-B7-H3-FITC mAb) to control the antitumor activity of anti-FITC CAR T cells. The effector functions of anti-FITC CAR T cells against OS, measured in vitro by tumor cell killing activity and cytokine production, are dependent on the presence of the anti-B7-H3-FITC mAb switch. Moreover, OS cells stimulate anti-FITC CAR T cells migration. In vivo, anti-B7-H3 mAb penetrates in the tumor and binds 143B OS tumor cells. Furthermore, anti-FITC CAR T cells reach tumor region and exert antitumor effect in an OS NSG mouse model only in the presence of the switch molecule. We demonstrate that anti-B7-H3-FITC mAb redirects the cytotoxic activity of anti-FITC CAR T cells against OS tumors suggesting that switchable CAR T cell platforms might be a plausible strategy against OS.

Identification of small extracellular vesicle protein biomarkers for pediatric Ewing Sarcoma.

Ewing Sarcoma (EWS) is the second most common osseous malignancy in children and young adults after osteosarcoma, while it is the fifth common osseous malignancy within adult age population. The clinical presentation of EWS is quite often non-specific, with the most common symptoms at presentation consisting of pain, swelling or general discomfort. The dearth of clinically relevant diagnostic or predictive biomarkers continues to remain a pressing clinical challenge. Identification of tumor specific biomarkers can lend towards an early diagnosis, expedited initiation of therapy, monitoring of therapeutic response, and early detection of recurrence of disease. We carried-out a complex analysis of cell lines and cell line derived small extracellular vesicles (sEVs) using label-free-based Quantitative Proteomic Profiling with an intent to determine shared and distinct features of these tumor cells and their respective sEVs. We analyzed EWS cells with different EWS-ETS fusions (EWS-FLI1 type I, II, and III and EWS-ERG) and their corresponding sEVs. Non-EWS controls included osteosarcoma, rhabdomyosarcoma, and benign cells, i.e., osteoid osteoma and mesenchymal stem cells. Proteomic profiling identified new shared markers between cells and their corresponding cell-derived sEVs and markers which were exclusively enriched in EWS-derived sEVs. These exo-biomarkers identified were validated by in silico approaches of publicly available protein databases and by capillary electrophoresis based western analysis (Wes). Here, we identified a protein biomarker named UGT3A2 and found its expression highly specific to EWS cells and their sEVs compared to control samples. Clinical validation of UGT3A2 expression in patient tumor tissues and plasma derived sEV samples demonstrated its specificity to EWS, indicating its potential as a EWS biomarker.

The HNRNPF/H RNA binding proteins and disease.

The members of the HNRNPF/H family of heterogeneous nuclear RNA proteins-HNRNPF, HNRNPH1, HNRNPH2, HNRNPH3, and GRSF1, are critical regulators of RNA maturation. Documented functions of these proteins include regulating splicing, particularly alternative splicing, 5' capping and 3' polyadenylation of RNAs, and RNA export. The assignment of these proteins to the HNRNPF/H protein family members relates to differences in the amino acid composition of their RNA recognition motifs, which differ from those of other RNA binding proteins (RBPs). HNRNPF/H proteins typically bind RNA sequences enriched with guanine (G) residues, including sequences that, in the presence of a cation, have the potential to form higher-order G-quadruplex structures. The need to further investigate members of the HNRNPF/H family of RBPs has intensified with the recent descriptions of their involvement in several disease states, including the pediatric tumor Ewing sarcoma and the hematological malignancy mantle cell lymphoma; newly described groups of developmental syndromes; and neuronal-related disorders, including addictive behavior. Here, to foster the study of the HNRNPF/H family of RBPs, we discuss features of the genes encoding these proteins, their structures and functions, and emerging contributions to disease. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > Splicing Regulation/Alternative Splicing RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Perinucleolar Compartment (PNC) Prevalence as an Independent Prognostic Factor in Pediatric Ewing Sarcoma: A Multi-Institutional Study.

The perinucleolar compartment (PNC) is a small nuclear body that plays important role in tumorigenesis. PNC prevalence correlates with poor prognosis and cancer metastasis. Its expression in pediatric Ewing sarcoma (EWS) has not previously been documented. In this study, we analyzed 40 EWS tumor cases from Caucasian and Hispanic patients for PNC prevalence by immunohistochemical detection of polypyrimidine tract binding protein and correlated the prevalence with dysregulated microRNA profiles. EWS cases showed staining ranging from 0 to 100%, which were categorized as diffuse (≥77%, n = 9, high PNC) or not diffuse (<77%, n = 31) for low PNC. High PNC prevalence was significantly higher in Hispanic patients from the US (n = 6, p = 0.017) and in patients who relapsed with metastatic disease (n = 4; p = 0.011). High PNC was associated with significantly shorter disease-free survival and early recurrence compared to those with low PNC. Using NanoString digital profiling, high PNC tumors revealed upregulation of eight and downregulation of 18 microRNAs. Of these, miR-320d and miR-29c-3p had the most significant differential expression in tumors with high PNC. In conclusion, this is the first study that demonstrates the presence of PNC in EWS, reflecting its utility as a predictive biomarker associated with tumor metastasis, specific microRNA profile, Hispanic ethnic origin, and poor prognosis.

The potential of PARP as a therapeutic target across pediatric solid malignancies

BackgroundPediatric cancer is the leading cause of disease-related death in children and the need for better therapeutic options remains urgent. Due to the limited number of patients, target and drug development for pediatrics is often supplemented by data from studies focused on adult cancers. Recent evidence shows that pediatric cancers possess different vulnerabilities that should be explored independently from adult cancers.MethodsUsing the publicly available Genomics of Drug Sensitivity in Cancer database, we explore therapeutic targets and biomarkers specific to the pediatric solid malignancies Ewing sarcoma, medulloblastoma, neuroblastoma, osteosarcoma, and rhabdomyosarcoma. Results are validated using cell viability assays and high-throughput drug screens are used to identify synergistic combinations.ResultsUsing published drug screening data, PARP is identified as a drug target of interest across multiple different pediatric malignancies. We validate these findings, and we show that efficacy can be improved when combined with conventional chemotherapeutics, namely topoisomerase inhibitors. Additionally, using gene set enrichment analysis, we identify ribosome biogenesis as a potential biomarker for PARP inhibition in pediatric cancer cell lines.ConclusionCollectively, our results provide evidence to support the further development of PARP inhibition and the combination with TOP1 inhibition as a therapeutic approach in solid pediatric malignancies. Additionally, we propose ribosome biogenesis as a component to PARP inhibitor sensitivity that should be further investigated to help maximize the potential utility of PARP inhibition and combinations across pediatric solid malignancies.

Abstract 3532: Activator protein-1 (AP-1) signaling inhibits the growth of Ewing sarcoma cells in response to DNA replication stress

Abstract Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the synthesis of deoxyribonucleoside triphosphates (dNTPs) and is required for DNA replication and the repair of DNA damage. Multiple types of cancer, including Ewing sarcoma tumors, are sensitive to RNR inhibitors or a reduction in the levels of either the RRM1 or RRM2 subunits of RNR. However, the polypharmacology and off-target effects of RNR inhibitors has complicated the identification of the precise mechanisms that regulate sensitivity and resistance to this class of drugs. Consequently, we used a conditional knockout (CRISPR/Cas9) and rescue approach to target RRM1 in Ewing sarcoma cells and identified that loss of RRM1 results in upregulation of the expression of multiple members of the activator protein-1 (AP-1) transcription factor complex, including c-Jun and c-Fos. This upregulation of AP-1 is mediated, in part, by SLFN11, which is expressed at high levels in Ewing sarcoma tumor and other pediatric sarcomas. Notably, the inducible expression of c-Jun and c-Fos in Ewing sarcoma cells impairs cell growth, downregulates the expression of the stem cell factor Sox2, and upregulates markers of cellular differentiation. In addition, small-molecule inhibitors of RNR, including gemcitabine and hydroxyurea, and HDAC inhibitors, which reduce the level of the RRM1 protein, also activate this AP-1 signaling pathway in Ewing sarcoma cells. Overall, these results provide novel insight into the critical pathways activated by loss of RNR activity and the mechanisms of action of inhibitors of RNR. Citation Format: David Gordon, Emma Croushore, Stacia Koppenhafer, Kelli Goss, Elizabeth Geary. Activator protein-1 (AP-1) signaling inhibits the growth of Ewing sarcoma cells in response to DNA replication stress. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3532.

Abstract 1706: A non-canonical MiT/TFE-dependent NRF2 program is a druggable vulnerability in multiple cancer types

Abstract The transcription factor NRF2 is a master regulator of cellular responses to oxidative stress, contributing to the pathogenesis of autoimmunity, metabolic disorders, and neurodegeneration. Somatic alterations in the NRF2 pathway also contribute to the growth and metastasis of many cancer types including ~30% of lung cancers. Still, the activation of NRF2 has frequently been observed in the absence of known genomic alterations, indicating that other pathways may drive its dysregulation. Further, approaches to target NRF2 pharmacologically have remained elusive. Here, we conducted a screen that identified a small molecule, ML329, exhibiting selective cytotoxicity in cells exhibiting NRF2 dependency and synthetic lethality to NRF2 pathway mutations across 489 cell lines. Surprisingly, we find that melanomas—which rarely have somatic mutations in the NRF2 pathway—were commonly sensitive to ML329. Melanomas were seen to exhibit NRF2-dependent metabolomic and transcriptional programs through the transcriptional activation of the adaptor protein p62/SQSTM1 by the melanocyte master regulator and oncoprotein MITF. This pathway was found to be conserved among all cancers characterized by genomic alterations of the MiT family (MITF, TFEB and TFE3) including subsets of renal cell carcinomas, pediatric sarcomas, and uveal and cutaneous melanomas. Our data identify a previously unrecognized, non-canonical mechanism of NRF2 activation by the MiT family, clarifying the regulation of NRF2 in pathologic and physiologic contexts. Pharmacologic inhibition of NRF2 could be valuable in the treatment of conditions with MITF family dysregulation. Citation Format: Xinbo Luo, Bart Lutterbach, Priya Pancholi, Yeon Sook Choi, Xiao Liu, Phillip Munson, Saqib Faisal, David A. Whipple, Robert A. Smith, Warren S. Weiner, David K. Johnson, Myriam Boukhali, Nicole S. Persky, Matthew G. Rees, Shunsuke Kitajima, David Barbie, Anuradha Roy, Michael Baltezor, Lian Rajewski, William McGuinness, John Haslam, Ananthan Sadagopan, Charles H. Yoon, Cory M. Johannessen, Christine G. Lian, Jason L. Hornick, Srinivas R. Viswanathan, David Liu, Vicki Nienaber, Wilhelm Haas, Frank J. Schoenen, David E. Fisher, Rizwan Haq. A non-canonical MiT/TFE-dependent NRF2 program is a druggable vulnerability in multiple cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1706.

Abstract 5240: Validation of research cohort based penetrance estimates for multiple cancer types and multiple primary cancers on clinically ascertained families

Abstract Age-at-onset penetrance is the probability of developing disease by a certain age given a patient’s characteristics. Accurate penetrance estimates are important for cancer screening and diagnosis, and for policy making in public health. We developed two Bayesian semi-parametric models: a model that estimates cancer-specific penetrances to the first primary (CS), and a model that estimates penetrances to the second primary without distinguishing between the cancer types (MPC). Model training requires a dataset that is enriched with enough MPC cases, as well as with competing risks from a variety of cancer types. Thus, we trained our models using data from families affected by the Li-Fraumeni Syndrome (LFS), a genetic disorder characterized by germline mutation in the gene TP53. This dataset consists of 189 families, all of which were ascertained via probands who were diagnosed with pediatric sarcoma at MD Anderson Cancer Center (MDACC) between 1944 and 1983. We have validated our penetrance estimates on independent LFS datasets with different ascertainment criteria, namely a patient cohort prospectively collected from high-risk clinics at MDACC, and another cohort from the National Cancer Institute (NCI). These datasets were meticulously collected for research purposes. However, clinical datasets, which resemble the data that genetic counselors encounter in counseling sessions, are severely impacted by missing data, most commonly missing ages at diagnosis and ages at last contact, and only represent a snapshot of family history without extended follow-up. To investigate the utility of our research-based risk prediction models in real-world clinics, we evaluated our penetrance estimates on clinically ascertained LFS families that were collected through the Clinical Cancer Genetics (CCG) program at MDACC in the past 10 years. The CCG dataset consists of 3,275 individuals across 124 families, of which 645 were diagnosed with at least one primary and 127 tested positive for TP53 mutation. Our CS model showed good performance when making cancer-specific predictions of the first primary, with Areas Under the Curve (AUCs) of 0.76, 0.81, and 0.68 for breast cancer, sarcoma, and all other cancer types combined. Our MPC model showed an AUC of 0.7 when predicting patients with MPC versus those with a single primary. Both the CS and MPC models perform better than the Chompret criteria when predicting TP53 mutation, with AUCs of 0.75 and 0.77 respectively. These results suggest that our models can be utilized for personalized risk predictions in clinical settings, despite being trained on a research-based dataset. Given that the Chompret criteria tends to predict TP53 mutation more conservatively, the ability of our models to cast a wider net is especially meaningful in the identification of potential carriers, which assists efficient genetic testing and screening. Citation Format: Nam H. Nguyen, Elissa B. Dodd-Eaton, Jessica L. Corredor, Jacynda Woodman-Ross, Nathaniel D. Hernandez, Angelica M. Gutierrez Barrera, Banu K. Arun, Wenyi Wang. Validation of research cohort based penetrance estimates for multiple cancer types and multiple primary cancers on clinically ascertained families. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5240.

Abstract 3534: Interrogating the immune microenvironment of a novel mouse model of fusion positive rhabdomyosarcoma

Abstract Immunotherapies have been largely ineffective for pediatric soft tissue sarcomas, in particular for fusion-positive rhabdomyosarcoma (FP-RMS), characterized by the PAX-FOXO1 gene fusion. We are hampered by a paucity of predictive, immune-competent mouse models of this cancer with which to develop novel and effective immune-based therapies. To overcome this barrier, we analyzed the immune microenvironment of one of the few genetically-engineered mouse models (GEMM) of FP-RMS. Briefly, this model is driven by conditional knock-in expression of Pax3:Foxo1 at the endogenous Pax3 locus, with or without conditional loss of MST1/2 (gene name Stk4/3), a Hippo pathway mediator that is modulated by PAX3-FOXO1. These two genotypes demonstrate different disease penetrance and tumor location, with mice bearing loss of MST1/2 exhibiting higher tumor penetrance and favoring head and neck disease sites. We hypothesize that these models exhibit differences in tumor-immune interactions in the microenvironment that correlate with these phenotypic differences. We aim to characterize the immune microenvironment differences between these tumors and to establish this GEMM as a predictive, syngeneic model of FP-RMS. Tumors were generated spontaneously from a GEMM of FP-RMS. Tumors expressed PAX3-FOXO1 and either carried a conditional loss of MST1/2 (MSTnull) or native MST1/2 (MSTwt). We analyzed 8 formalin-fixed, paraffin-embedded (FFPE) tumors and 2 tumor-derived cell lines from these novel models. FFPE tumor samples were analyzed via immunohistochemistry (IHC) for CD3 (T cell marker) and CD11b (myeloid cell marker) and were scored for immune cell infiltration. Overall, tumors demonstrate poor CD3+ T cell infiltration with significantly higher infiltration of CD11b+ myeloid cells (p=0.0002); this trend is also seen within the individual MSTwt (p=0.0286) and MSTnull cohorts (p=0.0286). There was no significant difference in CD3+ T cell or CD11b+ myeloid cell density between the two genotypes. The drivers of this immune phenotype and the role of Hippo pathway signaling in mediating RMS tumor immune cell infiltration warrants further investigation. Additional ongoing studies are utilizing a spatial gene expression platform to visualize the spatially-preserved transcriptome of intact tumors to reveal potential pathways mediating the immune microenvironment. Citation Format: Erin E. Resch, Kristianne M. Oristian, Elizabeth Mendes, Corinne M. Linardic, Brian H. Ladle. Interrogating the immune microenvironment of a novel mouse model of fusion positive rhabdomyosarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3534.

Abstract 3251: The immunostimulatory effect of 9-ING-41, a small molecule GSK-3 inhibitor, in sarcomas

Abstract Due to the rarity and genomic disparity of soft tissue and bone sarcomas, actionable therapeutic targets have been elusive. However, inhibition of GSK-3β has emerged as a potentially promising therapy that could be of great benefit to pediatric and adult sarcoma patients. Saos-2 osteosarcoma and 93T449 liposarcoma cell lines were pretreated at IC5072 with small molecule GSK-3β inhibitor 9-ING-41 (elraglusib). The cells were harvested for western blot analysis after 48 hours of treatment. Additionally, both cancer cell lines as well as TALL-104 T-cells and NK-92 NK cells were treated with 0.5 μM 9-ING-41 for 24 hours and harvested for Luminex cytokine profiling. The western blots demonstrated an increase in cPARP, an apoptotic marker, in both cancer cell lines after 9-ING-41 treatment. Additionally, an increase in PD-L1 expression was observed. The cytokine analysis revealed stimulation of immune cell activity in response to 9-ING-41. Treated T-cells had an increase in CXCL11, which is associated with T-cell recruitment, as well as an increased level of IL-18, which is shown to induce increased IFN-γ in Th1 cells. Additionally, NK-92 cells demonstrated an increase in IL-8 chemokine and an increase in soluble TRAIL (TRAIL/TNFSF10). The cancer cell lines showed a homogenous increase in growth factor TGF-ɑ, however, only the Saos-2 osteosarcoma cell line demonstrated an increase of IL-6. We are further validating the data with follow-up cytokine profiling. The increase in immunostimulatory cytokines as well as the increased expression of PD-L1 suggest a rationale for combining 9-ING-41 with immune checkpoint blockade therapy. The potential synergistic effect of these two therapies is currently under investigation with co-culture experiments of sarcoma and immune cells. The treatment cohorts for these experiments include 9-ING-41 combined with either anti-PD-L1, anti-PD-1, or anti-CTLA-4 immune checkpoint inhibitors. Our results suggest a promising combination therapeutic strategy for patients with soft tissue and bone sarcomas and future work will strive to better elucidate the mechanisms of efficacy. Citation Format: William J. MacDonald, Samuel Lew, Kelsey E. Huntington, Rishi R. Lulla, Bradley D. DeNardo, Wafik S. El-Deiry. The immunostimulatory effect of 9-ING-41, a small molecule GSK-3 inhibitor, in sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3251.